Colony-stimulating factors (CSFS) are cytokines that regulate the proliferation and differentiation of hematopoietic progenitor cells and the function of mature blood cells. Among CFSs, human granulocyte colony-stimulating factor (G-CSF) is thought to be a major stimulator of production of human neutrophilic granulocytes.
G-CSF is a glycoprotein produced in mononuclear cells, fibroblasts and endothelial cells. Purified G-CSF stimulates neutrophilic granulocyte colony formation from bone marrow progenitor cells, induces terminal differentiation (Nicola et al., JBC, Vol. 258, No. 14:9017-23, 1983), and suppresses self-replication and proliferation of leukocytes (Metcalf and Nicola, Leukemia Research, Vol. 9, 1:35-50, 1985). Human G-CSF (hG-CSF) was first purified from the human squamous carcinoma cell line CHU-2 (Nomura et al., EMBO J., Vol. 5, No. 5:871-76, 1986) and then from the human bladder carcinoma cell line 5637 (Welte et al., Proc. Narl. Acad. Sci. U.S.A., 82:1526-30, 1985; and Strife et al., Blood, Vol. 69, No. 5:1508-1523, 1987), in which the cell lines activity to promote human granulocyte colony formation was detected. The purified hG-CSF was identified to have a molecular weight of 18,000-19,000 Da and a pI value of 6.1 (the pI value varies from 5.5 to 6.1 according to the degree of glycosylation (Nomura et al., EMBO J., Vol.5, No. 5:871-76, 1986).
Neutrophilic granulocytes produced by stimulation of G-CSF are 10-20 μm in diameter, occupy over 70% of leukocytes, and play an important role in the protection of mammals from bacterial infection. However, owing to its half-life being shorter than macrophages and mononuclear cells, neutrophilic granulocytes must be produced continuously from pluripotent stem cells in bone marrow.
The hG-CSF isolated from human bladder carcinoma 5637 cells initially called pluripotent colony-stimulating factor or GM-CSF, based on the finding that it stimulates production of erythrocytes, megakaryocytes and macrophages in addition to neutrophilic granulocytes (Welte et al., Proc. Narl. Acad. Sci. U.S.A., 82:1526-30, 1985; Platzer et al., J. Exp Med, 162:1788-1801, 1985; and Nicola et al., Nature, 314:625-28, 1985), and affects pluripotent progenitor cells, for example, inducing proliferation of the human myeloid leukemia cell line HL-60 and the murine myelomonocytic leukemia cell line WEHI-3B(D+). However, in the studies excluding bone marrow cells and lymphocytes, the isolated protein was found to strongly stimulate neutrophilic granulocyte colony formation, and thus was assigned the nomenclature G-CSF (Welte et al., Proc. Narl. Acad. Sci. U.S.A., 82:1526-30, 1985; Metcalf, Science, 229:16-22, 1985; Metcalf, Blood, Vol. 67, No. 2:257-67, 1986; Metcalf, Proc. R. Soc. Lond. B., 230:389-423, 1987; and Sachs, Science, 238:1374-79, 1987). When treating with the isolated hG-CSF a mixture of hematopoietic colony-forming progenitor cells derived from human bone marrow cells lacking adherent cells and T-lymphocytes, neutrophilic granulocyte colony formation was observed after 7 days (Welte et al., Proc. Narl. Acad. Sci. U.S.A., 82:1526-30, 1985; and Platzer et al., J., Exp. Med., 162:1788-1801, 1985). In addition, the isolated hG-CSF stimulates differentiation of WEHI-3B(D+) cells.
Murine G-CSF (mG-CSF) has biological activity similar to hG-CSF. That is, mG-CSF produces neutrophilic granulocyte colonies in the CFU-GM assay, and induces terminal differentiation of WEHI-3B(D+) cells. In addition, hG-CSF functions not only in human bone marrow cells but also in murine bone marrow cells. Conversely, mG-CSF acts on both human and murine bone marrow cells.
In animals administered with G-CSF, the in vivo effects of G-CSF are regulated by its administered amount, and, when G-CSF treatment is stopped, the blood level of neutrophilic granulocytes is maintained at normal levels. However, the blood levels of G-CSF receptor-lacking blood cells, that is, erythrocytes, mononuclear cells and lymphocytes, are not changed.
In bone marrow cells and splenocytes, the G-CSF receptor is essential for differentiation of myeloid precursor cells into neutrophilic granulocytes. Also, the fact that mature neutrophilic granulocytes carry the G-CSF receptor suggests that G-CSF activates the mature cells. Receptor numbers are between 50 and 500 per cell. The concentration of G-CSF required for half-maximal stimulation is about 10 pM, while G-CSF has an equilibrium dissociation constant (Kd) of about 60-80 pM for G-CSF receptor binding. This fact indicates that the proliferation induced by G-CSF occurs in the presence of low levels of G-CSF receptors.
In addition to mature neutrophils, G-CSF affects neutrophil progenitor cells. That is, G-CSF enhances survival of mature neutrophils (Begley et al., Blood, Vol. 68, No. 1:162-66, 1986), and does not induce differentiation of the acute myeloblastic leukemia cells into mature cells, resulting in specific activation of neutrophils (Lopex et al., J. Immunol. Vol 131 No6:2983-2988, 1983; and Platzer et al., J. Exp. Med., 162:1788-1801, 1985).
In addition, when G-CSF was administered to an animal in which neutropenia had been induced by treatment with 5-fluorouracil and cyclophosphamide, proliferation of neutrophils was found to remarkably increase. In the clinical trials based on this finding, when G-CSF was administered into chemotheraphy-receiving malignant tumor patients (Bronchud et al., Br. J. Cancer, 56:809-13, 1987; Gabrilove et al., New England J. Med., Vol. 318, No. 22:1414-22, 1988; and Morstyn et al., Lancet, March 26:667-71, 1988), and patients undergoing bone marrow cell transplantation after treatment with radioisotopes of cyclophosphamide (Kodo et al., Lancet, July 2:38-39, 1988), patients feel only slight pain and G-CSF rarely causes side effects and induces an increase of neutrophilic granulocytes in both cases. These results indicate that G-CSF administration helps chemotheraphy-received of bone marrow-transplanted patients, protecting them from bacterial or fungal infection occurring when recovery of the neutrophilic granulocyte levels to the normal levels is delayed. Such successful clinical trials allow G-CSF to be applied to a variety of patients suffering from neutropeina.
Molecular and genetic properties of G-CSF were identified by recombinant DNA technology (Clark and Kamen, Science, 236:1229-37, 1987), and a variety of studies of the functions of G-CSF were performed in vivo and in vitro using recombinant G-CSF. Human G-CSF was cloned from a cDNA library constructed with mRNA prepared from CHU-2 cells and human bladder carcinoma 5637 cells (Nagata et al., Nature, 319:415-18, 1986; Nagata et al., EMBO J., Vol. 5, No. 3:575-81, 1986; and Souza et al., Science, 232:61-65, 1986). In this study, two different cDNAs for human G-CSF were isolated. The nucleotide sequence analysis of both cDNAs indicated that they encode polypepetides consisting of 207 and 204 amino acids, respectively, and their translated products have a presequence (a secretory leader sequence) of 30 amino acids at the N-terminus. Two polypeptides coded by these cDNAs are different at the 35th position where three amino acids (Val-Ser-Glu) are deleted/inserted. Therefore, mature G-CSF protein is composed of 174 amino acids (MW 18,671 Da) or 177 amino acids (MW 18,987 Da).
The 174 amino acid G-CSF has an over 20-fold higher activity than the other consisting of 177 amino acids. However, it is not still clear that the two different forms are expressed in the human body. Human G-CSF does not have the N-glycosylation sequence (Asn-X-Ser/Thr (N-X-S/T)), but has an O-glycosylation site at the Thr-133 position. When recombinant human G-CSF prepared using the cDNA for human G-CSF was produced in E. coli (Souza et al., Science, 232: 61-65, 1986; Delvin et al., Gene, 65: 13-22, 1988) and animal cells (Tsuchiya et al., EMBO J., Vol. 6, No. 3: 611-16, 1987), recombinant human G-CSF produced in E. coli was found to have an activity identical to the natural form and the form expressed in animal cells. These results indicate that glycosylation is not critical essential for G-CSF activity. Human G-CSF ha no amino acid sequence homology with GM-CSF, interleukin-3 and M-CSF, and possesses two disulfide bonds (Cys36-Cys42 and Cys64-Cys74) formed by 4 of 5 cystein residues.
Due to their low in vivo stability, most of the physiologically active proteins used as drugs are excessively or frequently administered to patients in order to maintain an appropriate concentration capable of offering satisfactory therapeutic effects. This administration pattern causes pain in patients and inconvenience. Therefore, there is a need for development of physiologically active proteins having improved in vivo stability and thus resolving the problems in the prior art.
In order to improve in vivo stability of physiologically active proteins, interferon-alpha can be conjugated with polyethylene glycol as disclosed in International Pat. Publication No. WO9848840, and human growth hormone can be microcapsulated as disclosed in U.S. Pat. No. 6,399,103. However, these methods are disadvantageous in that additional steps should be carried out after production in a microorganism and purification of a target protein. In addition, cross-linkage can be formed at undesired positions. Moreover, the manufacturing processes do not ensure homogeneity of final products.
Another approach uses glycosylation. Cell surface proteins and secretory proteins produced in eukaryotic cells are modified by glycosylation. Glycosyl modification is know addition to their physiological properties.